Role of GPR54 Signaling in Pubertal Disorders Summary/Abstract The long term goal of this project is to identify factors that regulate the timing of pubertal onset and reproductive maturation. The identification of GPR54, a G-protein coupled receptor, and its ligand, kisspeptin, as upstream regulators of GnRH secretion has led to intense research to elucidate their roles in the regulation of the reproductive axis. Inactivating mutations in GPR54 cause failure to undergo puberty and infertility. In contrast, early stimulation of this receptor triggers precocious puberty in mice. Our preliminary results indicate that GPR54 is desensitized and internalized in response to continuous kisspeptin stimulation, and that a GPR54 amino acid substitution identified in a female patient with central precocious puberty (a disorder with disproportionately high female incidence) increases GPR54 responsiveness by delaying the desensitization of the receptor. Thus, we hypothesize that the timing of signaling and desensitization of GPR54 is critical for its role in controlling puberty and reproduction, and that amino acid substitutions in GPR54 may affect its responsiveness by interfering with signaling or desensitization, thereby contributing to the clinical presentation. Although G-protein coupled receptor desensitization is generally strongly regulated, no data have been published on GPR54 desensitization. The short term goal of this project is to define the mechanisms underlying GPR54 desensitization, in order to understand how genetic mutations of this receptor affect these mechanisms and hence the timing of pubertal onset and sexual maturation. Specifically, the aims of this proposal are to: (1) Define the mechanisms of GPR54 desensitization, focusing on the roles of phosphorylation and arrestin recruitment;(2) Define the mechanisms of GPR54 internalization, focusing on the roles of arrestin, dynamin, and clathrin;and (3) Define the fate of the internalized GPR54, to determine whether the receptor is directed to lysosomal degradation or recycled back to the plasma membrane. In each case, the effects of two mutations in GPR54, one identified in a patient with precocious puberty, and the other in a patient with hypogonadotropic hypogonadism, on these pathways will be determined. A thorough understanding of the mechanisms underlying GPR54 signaling may uncover the basis of gender differences in normal and abnormal pubertal development, as well as reveal a new array of potential targets of pharmacological manipulation for the treatment and prevention of abnormal pubertal development and possibly other reproductive disorders. Role of GPR54 Signaling in Pubertal Disorders Narrative The goal of this project is to define the mechanisms underlying the regulation of GPR54 receptor signaling and desensitization, in order to understand how genetic mutations of this receptor affect these mechanisms and hence the timing of pubertal onset and sexual maturation. These studies are expected to offer important contributions to our understanding of the mechanisms underlying the reproductive disorders in the patients carrying the mutations. These insights, in turn, may contribute to future development of therapies designed to modulate the timing of puberty by manipulating the kisspeptin-GPR54 signaling system.